The recruitment and trafficking of CD4+ cells in the body is modulated by chemoattractant proteins, typically produced in response to inflammatory stimuli. Reviewed in Taub (1996). Directed CD4+ cell movement up concentration gradients of chemoattractants results in trafficking of CD4+ cells out of the circulation, through the vascular endothelium, and into peripheral tissue to the site of highest localized concentration of chemoattractants. By secreting their own chemoattractants, CD4+ cells may direct the subsequent accumulation of various other cells types involved in host defense mechanisms, such as neutrophils. While the localized accumulation of CD4+ cells may be important for defense against infection, accumulation of CD4+ cells can also be part of a chain of events resulting in an unwanted inflammatory response.
For example, infiltration and accumulation of CD4+ cells in the respiratory tract helps mediate clearance of Cryptococcus neoformans infections, which otherwise will disseminate to cause meningitis. Huffnagle et al. (1995). By contrast, uncontrolled infiltration and activation of CD4+ cells in response to bacterial antigens appears to be the underlying cause of inflammatory bowel disease. Van Deventer et al. (1997). Localized CD4+ cell accumulation has been implicated specifically in response to infections and inflammatory conditions, including inflammatory bowl disease, bacterial infection, viral infection, atherosclerosis, asthma, graft versus host disease (GVHD), endotoxemia, uveoretinitis, psoriasis, and granulomatous diseases. Center (1996); Jinquan et al. (1995); Taub (1996); Van de Kerkhof et al. (1996). See, in general, Staniford (1997).
Another disease associated with CD4+ cell physiology is HIV infection. A defining feature of progression of HIV infection to Acquired Immune Deficiency Syndrome (AIDS) is a decline in the number of CD4+ cells in the infected individual. Reviewed in Levy (1993). Disease progression also strongly correlates with a switch from non-syncytium-forming (NSI) HIV variants to syncytium-forming (SI) variants within infected individuals. Richman et al. (1994); Miedema (1992). For example, a recent study found that the adenoid tissue of 13 of 13 asymptomatic individuals contained syncytia formed apparently from fusion of dendritic cells and T cells. Frankel et al. (1996).
Recent studies have provided a potential link between CD4+ cell loss and syncytium formation by implicating CD4+ cell chemotaxis in the propagation of syncytia. Syncytia have been found to release CD4+ cell chemoattractants, potentially providing a mechanism by which CD4+ cells can be recruited throughout the body to their demise by incorporation into short-lived syncytia. Shutt et al. (1998).
Syncytia are multinucleated conglomerates of HIV-infected cells having up to many thousands of times the volume of a single cell. They are formed when the virally encoded glycoprotein gp120 on the surface of infected cells interacts with the CD4 receptor of uninfected cells to initiate cell fusion. Kowolski et al. (1987); McDougal et al. (1986); Lifson et al. (1986a,b). Syncytia also are phagocytotic and may engulf entire CD4+ cells. Sylwester et al. (1995). In vitro studies have indicated that up to 90% of T cell death is accounted for by their incorporation into syncytia. Vast quantities of HIV are released upon the death of syncytia, which infect non-fused CD4+ cells, thus creating a self-perpetuating cycle of infection and CD4+ cell death. Sylwester et al. (1997).
In addition to perpetuating HIV infection, syncytia can directly cause tissue destruction by virtue of motile properties retained from constituent lymphocyte cells. Soll, 1997. Syncytia move by extending and retracting giant pseudopodia and filopodia, which penetrate and disrupt collagen and endothelial tissue substrates. Sylwester et al. (1998). The ability of syncytia to degrade and extravasate through endothelial tissue may be related to the destruction of lymph node architecture and the leakiness of blood vessels in an individual bearing the syncytium-inducing variant of HIV. This property may also account for the apparent absence of syncytia in blood vessels, since these large cells would likely become stuck in capillaries, where they could then extravasate into the periphery.
In video recordings of fields of single cells and syncytia in the act of fusing in vitro, it was apparent that single cells and small syncytia moved in a persistent and directed fashion towards large syncytia, suggesting that the latter released a T cell chemoattractant. To confirm this hypothesis, a specialized single-cell chemotaxis chamber was used to discriminate chemokinesis from chemotaxis. Shutt et al. (1998). Chemokinesis is accelerated, non-vectorial movement, while chemotaxis is directed movement of cells up a concentration gradient of a chemoattractant. The microfilter assay, first introduced by Boyden (1962), was not used because it cannot unambiguously distinguish chemotaxis form chemokinesis, even when the appropriate corrections are performed. Wilkinson (1988); Zigmond (1978); Zigmond et al. (1973); Rhodes (1982); Shutt et al. (1998).
Syncytia release two chemotactic components into the supernatant, having approximate molecular weights of 30 and 120 kDa. Shutt et al. (1998). Virally encoded glycoprotein gp120 is released into the medium by HIV-infected cells, implicating the 120 kDa chemoattractant as gp120. Gelderblom et al. (1985); Schneider et al. (1986). This was confirmed by the ability of anti-gp120 antibody to block the high molecular weight chemoattractant, and by the ability of purified gp120 to attract T cells. Shutt et al. (1998).
The identity of the lower molecular weight component remained unknown. HIV virus encodes several low molecular weight proteins, including Rev, p24, Nef (27 kDa), a negative regulator of viral replication, and Tat (15.5 kDa), an activator of viral gene expression, but there was no suggestion in the art that these proteins function as chemoattractants, with the possible exception of Tat. Tat induces the migration of monocyte-derived dendritic cells and monocytes across a membrane in a Boyden chamber in a concentration-dependent manner. Benelli et al. (1998). While these authors concluded that Tat acted as a dendritic cell chemoattractant, the absence of critical controls suggested by Zigmond et al. (1973) for the microfilter assay complicates the distinction between a chemotactic and a chemokinetic effect.
There have been, however, no reports of either chemotactic or chemokinetic activity by the other low molecular weight HIV proteins. Although interactions of Nef with various cellular proteins have been characterized, the known interactions are not suggestive of chemotactic or chemokinetic activity. The activity of a putative CD4+ cell chemoattractant, interleukin-16, is thought to require coupling between CD4 and the cellular kinase p56lck. Ryan et al. (1995). However, intracellular Nef expression has been demonstrated to reduce cell surface expression of CD4. Gratton et al. (1996). Further, Nef apparently disrupts the interaction between CD4 and p56lck, either directly or, more likely, indirectly through other proteins recruited by Nef. Otake et al. (1994); Benichou et al. (1994).
Thus, there is a need in the art to modulate CD4+ cell chemotaxis. Such a capability will enhance treatment of a disease associated with localized CD4+ cell accumulation. It is desirable to direct CD4+ chemotaxis to a localized site of infection in circumstances where CD4+ cell intervention is desired for a favorable clinical outcome. Likewise, it is desirable to prevent CD4+ cell chemotaxis at sites where this accumulation leads to undesired inflammatory reactions. Alternately, it is desirable to redirect CD4+ cell accumulation to sites where their accumulation will be less detrimental.
Modulation of CD4+ cell chemotaxis would be desirable in treatment of HIV infection as a means of inhibiting the syncytium-induced chemotaxis of CD4+ cells out of circulation and into peripheral or lymphoid tissue, where they may fuse with or be engulfed and destroyed by syncytia. By inhibiting syncytium-mediated T cell death, the progression of HIV infection into AIDS may be retarded.